CN105309314B - A kind of method that anaesthetic Dracocephalum moldavica Tissue Culture Regeneration System is set up - Google Patents
A kind of method that anaesthetic Dracocephalum moldavica Tissue Culture Regeneration System is set up Download PDFInfo
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Abstract
The present invention relates to a kind of induction of anaesthetic Dracocephalum moldavica aseptic seedling Cotyledon Callus and the method for Regeneration System, belong to biological technical field Plant Tissue Breeding category.Including following steps：(1) acquisition of Dracocephalum moldavica aseptic seedling；(2) induction of Cotyledon Callus and squamous subculture；(3) the induction differentiation culture of adventitious bud；(4) the rooting induction culture of regeneration plant；(5) domestication and transplanting of Dracocephalum moldavica regeneration plant.This method is explant from the cotyledon of Dracocephalum moldavica aseptic seedling, the species and concentration of specific hormone are added by adjusting, the improvement of distinct methods is carried out to MS culture mediums, the callus of anaesthetic Dracocephalum moldavica is obtained eventually through induction and then establishes regenerating system, obtain explant easily, operating method is relatively easy, and cultivation cycle is short, breeding coefficient is high, provides good seedling or the genetic transformations etc. of the species can be carried out as outstanding scientific research material further to expand cultivation Dracocephalum moldavica and study.
Description
Technical field：
The invention belongs to biological technical field method for plant tissue culture, and in particular to anaesthetic Dracocephalum moldavica tissue cultures regenerate
The method for building up of system.
Background technology：
Dracocephalum moldavica (Dracocephalum moldavica L.), alias rubs eye, mountain mint, Lan Qiuhua, teosinte, perfume (or spice)
Beggar, etc..Its quality standard is recorded in Inner Mongol mongolian medicine standard, the Sanitation Ministry medicine standard dimension medicine fascicle respectively, is also once recorded
In version Chinese Pharmacopoeia in 1977, Chun Xing sections green grass or young crops Cymbidium annual herb plant, medicinal part is ground herb, is distributed mainly on
China North China, northeast, particularly northwest China area, the Inner Mongol and Xinjiang.The entitled Bi Yanggu of Mongolia, is that great exploitation is latent
The natural medicinal plant of power and national characters, with allevating angina pectoris, improves myocardial ischemia, reduction blood viscosity and platelet aggregation
The effects such as rate, acts on.In addition to as Chinese herbal medicine, Dracocephalum moldavica is because containing substantial amounts of volatile oil, being prepare lemonene system spices important
Raw material.In addition, the tender cauline leaf of Dracocephalum moldavica can be edible as special vegetable, dry cauline leaf and seed can make tea, flavoring or making
Spices.The fragrance that the multifunctional medical health that Dracocephalum moldavica is contained with it acts on and shown unique characteristics turns into a kind of great exploitation potential
Natural medicinal plant resource and Aromatic plant resources.
In recent years, with the propulsion of national " modernization of Chinese medicine and industrialization development " action, the exploitation to Dracocephalum moldavica
Obtained fast development, and the acquisition of raw material is excavated mainly by field, the protection of resources of wild Dracocephalum moldavica and foster, introduce a fine variety with
Domestication is in full swing.Dracocephalum moldavica is mainly by seminal propagation, but its seed is very small, and should not preserve easy devitalization.
Plant Tissue Breeding and the foundation of regenerating system are the important channels of quick breeding plant improved seeds detoxic seedling, and to Dracocephalum moldavica
The research for carrying out tissue cultures acquisition regeneration plant is not reported so far.Timely and effectively to protect with developing anaesthetic Dracocephalum moldavica,
Ensure its sustainable use, the present invention using the hypocotyl of Dracocephalum moldavica aseptic seedling as explant, by callus induction, differentiation,
Breed, take root, hardening, the serial procedures such as transplanting have successfully been obtained Dracocephalum moldavica regeneration plant, establishing tissue culture rapid propagation system, be
Dracocephalum moldavica Clone Breeding and genetic transformation, factorial praluction nursery meet the market demand and provided fundamental basis and practical basis.
The content of the invention：
The present invention provides a kind of method that anaesthetic Dracocephalum moldavica Tissue Culture Regeneration System is set up.
Technical solution
The method for building up of described Dracocephalum moldavica Cotyledon Callus regenerating system, comprises the following steps：
(1) the surface of the seed sterilization and Aseptic seedling culture：
Full seed, Dracocephalum moldavica seed of the same size are selected, rinsing 4-7 min through flowing water removes surface dirt debris,
On 4-7 rear placement superclean bench of aseptic water washing, 30-40 s, aseptic water washing 2-4 are soaked with the ethanol of volumetric concentration 70%
It is secondary；The HgCl of volumetric concentration 0.1% is used again24-6 min are sterilized, sterilized water concussion is rinsed 4-6 times, is seeded in containing 100 mL MS0
In the triangular flask of solid medium, triangular flask body is wrapped up with brown paper, cultivated under the conditions of 28 ± 2 DEG C.Seed is observed daily
Pollution and sprouting state.After removal brown paper illumination cultivation, illumination 12-14 h/d, intensity of illumination 1800- after seed sprouting
2200 1ux。
(2) induction of callus
The Dracocephalum moldavica aseptic seedling of the robust growth of step (1) culture is taken, the cotyledon for cutting aseptic seedlings is inoculated in induction training
Support base MS1On, every bottle of inoculation 3-4 block is transferred to after light culture 9-12 d under normal illumination and cultivated.Inducing culture MS1Using MS as base
Basal culture medium, addition 6-BA 0.5 mg/L, 2,4-D 1.0,0.5 mg/L of mg/L, NAA, the mg/L of sucrose 30, the mg/ of agar 10
Observed after L, pH 5.8-6.0,13-16 d and count callus induction rate.Induction illumination cultivation condition be：Illumination 12-14
H/d, intensity of illumination 1800-2200 1ux, 2 DEG C of 28 scholar of temperature.
(3) squamous subculture of callus
By step (2) obtained yellow green callus will be induced to be seeded in subculture medium MS2Middle progress subculture training
Support.Subculture medium MS2Using MS as minimal medium, addition 6-BA 0.5 mg/L, 2,4-D 0.5,0.5 mg/ of mg/L, NAA
L, the mg/L of sucrose 30, agar 10 mg/L, pH 5.8-6.0, squamous subculture condition is：Illumination 12-14 h/d, intensity of illumination
1800-2200 1ux, 2 DEG C of 28 scholar of temperature.
(4) the induction differentiation of adventitious bud
Trained adventitious buds differentiation is gone to by the yellow green callus obtained after step (3) continuous squamous subculture 3-4 time
Support base MS3It is middle to be induced, adventitious buds differentiation culture medium using MS as minimal medium, the mg/L of addition exogenous hormone 6-BA 0.8,
The mg/L of NAA 0.1, the mg/L of sucrose 30, agar 10 mg/L, pH 5.8-6.0, condition of culture is：Illumination 12-14 h/d, illumination
Intensity 1800-2200 1ux, 2 DEG C of 28 scholar of temperature.
(5) root induction
Root induction culture medium MS is transferred to when regeneration bud 2-3 pieces or so are obtained by step (4) adventitious bud inducing4Carry out
Rooting induction, rooting induction culture medium is using 1/2MS as minimal medium, the mg/L of addition exogenous hormone IBA 0.2, sucrose 30
Mg/L, agar 6 mg/L, pH 5.8-6.0, condition of culture is：Illumination 12-14 h/d, intensity of illumination 1800-2200 1ux, temperature
Spend 2 DEG C of 28 scholar.
(6) Transplantation of Regenerated Plantlets
By the rooting induction of 12-15d or so steps (5), regeneration plant is grown after 4-6 bar roots, and triangular flask bottleneck is beaten
Open, be placed under natural conditions（At shady and cool, air circulation）Hardening, regrowth is taken out, rushed through flowing water after 7-8 days from triangular flask
Wash the culture medium residue in foundation portion off, then, 6-8 is soaked with volumetric concentration 0.08-0.13% carbendazim with distilled water flushing several times
Min, goes in transplanting medium, and matrix is pine bark, peat soil, liver moss living and haydite（By 1：1：1：1 mixing）, outer button shading modeling
Material, pours permeable 1-3 times daily, cultivates 2 DEG C of 28 scholar of temperature, evening minimum temperature is not less than 20 DEG C.Treat that seedling grows 3-4 pieces new
Leaf, height of seedling 8-12cm or so is transplanted under normal soil to be continued to cultivate in natural environment.
The present invention also has following additional technical feature：
The MS0Solid medium is basic MS (Murashige and Skoog, 1962) culture medium, containing following dense
The material of degree：1.65 g/LNH4NO3、1.90 g/L KNO3、0.17 g/L KH2PO4、0.1807 g/L MgSO4·7H2O、
0.332 g/L CaCl2；Trace element：22.3 mg/L MgSO4·4H2O、6.2 mg/L N3BO3、8.6 mg/L ZnSO4·
7H2O、0.83 mg/L KI、0.25 g/L Na2MoO4·2H2O、0.025 mg/L CuSO4·7H2O、0.025 mg/L
CoCl·6H2O、27.8 mg/L FeSO4·7H2O、37.3 mg/L Na2-EDTA；Vitamin：The mg/L of inositol 100, nicotinic acid
0.5 mg/L, L- glycine 2 mg/L, VB6 0.5 mg/L, VB1 0.1 mg/L, sucrose 30 g/L, agar 8-10 g/L.
The inducing culture MS1For using MS as minimal medium, addition 6-BA 0.5 mg/L, the mg/L of 2,4-D 1.0,
The mg/L of NAA 0.5, the mg/L of sucrose 30, agar 10 mg/L, pH 5.8-6.0.
The subculture medium MS2For using MS as minimal medium, addition 6-BA 0.5 mg/L, the mg/L of 2,4-D 0.5,
The mg/L of NAA 0.5, the mg/L of sucrose 30, agar 10 mg/L, pH 5.8-6.0.
The adventitious buds differentiation culture medium MS3 be using MS as minimal medium, the mg/L of addition exogenous hormone 6-BA 0.8,
The mg/L of NAA 0.1, the mg/L of sucrose 30, agar 10 mg/L, pH 5.8-6.0.
The root media MS4Using 1/2MS as minimal medium, to add the mg/L of exogenous hormone IBA 0.2, sucrose
30 mg/L, agar 6 mg/L, pH 5.8-6.0.The 1/2MS nutrient media componentses include a great number of elements：0.825 g/LNH4NO3、
0.95 g/L KNO3、0.085 g/L KH2PO4、0.09035 g/L MgSO4·7H2O、0.166 g/L CaCl2；Micro member
Element：22.3 mg/L MgSO4·4H2O、6.2 mg/L N3BO3、8.6 mg/L ZnSO4·7H2O、0.83 mg/L KI、0.25
g/L Na2MoO4·2H2O、0.025 mg/L CuSO4·7H2O、0.025 mg/L CoCl·6H2O、27.8 mg/L
FeSO4·7H2O、37.3 mg/L Na2-EDTA；Vitamin, the mg/L of inositol 100, the mg/ of 0.5 mg/L, L- glycine of nicotinic acid 2
L、VB6 0.5 mg/L、VB1 0.1 mg/L。
The transplanting medium is that pine bark, peat soil, liver moss living and haydite press 1：1：1：1 mixing gained.
The normal soil is outdoor random acquired planting soil.
Dracocephalum moldavica is concentrated mainly on chemical composition as a kind of typical illiteracy, the conventional ethnic drug of dimension to its research carried out
Analysis in terms of setting up Dracocephalum moldavica regenerating system by tissue cultures with clinical practice, being not reported so far.Using the present invention's
Beneficial effect is to provide a kind of method that anaesthetic Dracocephalum moldavica Tissue Culture Regeneration System simple to operate is set up.Using Dracocephalum moldavica without
The cotyledon of vaccine carries out rising in value very fast after callus induction, callus squamous subculture as explant, and differentiation degree is high, warp
Survival rate is high after the aftergrowth obtained after culture of rootage is transplanted, and can obtain a large amount of Dracocephalum moldavicas in a short time using this method regenerates
Plant, provides good seedling or the something lost of the species can be carried out as outstanding scientific research material further to expand cultivation Dracocephalum moldavica
Pass the research such as conversion.
Brief description of the drawings：
The Dracocephalum moldavica aseptic seedling that Fig. 1 cultivates for the present invention；
Fig. 2 is the situation of Dracocephalum moldavica aseptic seedling Cotyledon culture culture 5 days of the present invention；
Fig. 3 is the situation that Dracocephalum moldavica cotyledon callus of the present invention is induced 20 days；
Fig. 4 is that Dracocephalum moldavica callus breaks up adventitious bud；
Fig. 5 is the differentiation of Dracocephalum moldavica adventitious root；
Fig. 6 is Dracocephalum moldavica Transplantation of Regenerated Plantlets.
Case is embodied：
The implementation to the present invention is described in further detail below：
A kind of method that anaesthetic Dracocephalum moldavica Tissue Culture Regeneration System is set up, the seed of plantation Dracocephalum moldavica aseptic seedling picks up from interior
Mongolian Xilinguole League grassland, it uses following steps：
Step one：Full seed, Dracocephalum moldavica seed of the same size are selected, rinsing 4-7 min through flowing water removes surface dirt
On native debris, 4-7 rear placement superclean bench of aseptic water washing, 30-40 s, sterilized water are soaked with the ethanol of volumetric concentration 70%
Rinse 2-4 times；The HgCl of volumetric concentration 0.1% is used again24-6 min are sterilized, sterilized water concussion is rinsed 4-6 times, is seeded in containing 100
mL MS0Solid medium（MS minimal mediums, pH5.8）Triangular flask in, triangular flask body is wrapped up with brown paper, 28 ± 2
Cultivated under the conditions of DEG C.Pollution and the sprouting state of seed are observed daily.Germination rate=(germination seed number/inoculation seed number) ×
100%, after removal brown paper illumination cultivation, illumination 12-14 h/d, intensity of illumination 1800-2200 1ux after seed sprouting.
The MS0Solid medium is with basic MS（Murashige and Skoog, 1962）Based on culture medium contain with
The material of lower mass-volume concentration：The g/L of sucrose 30, agar 8-10 g/L, 5.8-6.0 is adjusted to before sterilizing by pH.
The basic MS culture medium component includes a great number of elements：1.65 g/LNH4NO3、1.90 g/L KNO3、0.17 g/L
KH2PO4、0.1807 g/L MgSO4·7H2O、0.332 g/L CaCl2；Trace element：22.3 mg/L MgSO4·4H2O、
6.2 mg/L N3BO3、8.6 mg/L ZnSO4·7H2O、0.83 mg/L KI、0.25 g/L Na2MoO4·2H2O、0.025
mg/L CuSO4·7H2O、0.025 mg/L CoCl·6H2O、27.8 mg/L FeSO4·7H2O、37.3 mg/L Na2-
EDTA；Vitamin, the mg/L of inositol 100,0.5 mg/L, L- glycine of nicotinic acid 2,0.5 0.1 mg/ of mg/L, VB1 of mg/L, VB6
L。
Step 2：The Dracocephalum moldavica aseptic seedling of the robust growth of step (1) culture is taken, the cotyledon for cutting aseptic seedlings is inoculated in
Inducing culture MS1On, every bottle of inoculation 3-4 block is transferred to after light culture 8-10 d under normal illumination and cultivated.Observed after 12-15 d
And count callus induction rate.Inducing culturing condition is：Illumination 12-14 h/d, the 1ux of intensity of illumination 2000, the scholar 2 of temperature 28
℃.The inducing culture MS1Using MS as minimal medium, to add the mg/L of 6-BA 0.5,2,4-D 1.0 mg/L, NAA
0.5 mg/L, the mg/L of sucrose 30, agar 10 mg/L, pH 5.8-6.0.Inductivity=generation callus explant number/inoculation
Explant number, the average inductivity of 3 batch experiments is 91.2%, is shown in Table 1：
Step 3：By step (2) obtained faint yellow callus will be induced to be seeded in subculture medium MS2It is middle to carry out
Squamous subculture.Condition of culture is：Illumination 12-14 h/d, the 1ux of intensity of illumination 2000,2 DEG C of 28 scholar of temperature.The squamous subculture
Base MS2 is addition 6-BA 0.5 mg/L, 2,4-D 0.5,0.5 mg/L of mg/L, NAA, sucrose 30 using MS as minimal medium
Mg/L, agar 10 mg/L, pH 5.8-6.0.
Step 4：Step will be passed through（3）The faint yellow callus obtained after continuous squamous subculture 3-5 times goes to adventitious bud
Differential medium MS3The middle induction for carrying out adventitious bud, condition of culture is：Illumination 12-14 h/d, the 1ux of intensity of illumination 2000, temperature
Spend 2 DEG C of 28 scholar.The adventitious buds differentiation culture medium MS3Using MS as minimal medium, to add the mg/ of exogenous hormone 6-BA 0.8
The mg/L of L, NAA 0.1, the mg/L of sucrose 30, agar 10 mg/L, pH 5.8-6.0.The callus of differentiation rate=differentiate regeneration bud/
Induce the callus sum of differentiation.3 batch experiment adventitious bud average mark rates are 84.2%, are shown in Table 2：
Step 5：Root induction culture medium MS is transferred to when obtaining regeneration bud 2-4 pieces or so by adventitious bud inducing4Given birth to
Root induction, condition of culture is：Illumination 12-14 h/d, the 1ux of intensity of illumination 2000,2 DEG C of 28 scholar of temperature.The rooting induction training
Support base MS3Using 1/2MS as minimal medium, to add the mg/L of exogenous hormone IBA 0.2, the mg/L of sucrose 30, the mg/L of agar 6,
pH 5.8-6.0。
The basic 1/2MS nutrient media componentses include a great number of elements：0.825 g/LNH4NO3、0.95 g/L KNO3、
0.085 g/L KH2PO4、0.09035 g/L MgSO4·7H2O、0.166 g/L CaCl2；Trace element：22.3 mg/L
MgSO4·4H2O、6.2 mg/L N3BO3、8.6 mg/L ZnSO4·7H2O、0.83 mg/L KI、0.25 g/L Na2MoO4·
2H2O、0.025 mg/L CuSO4·7H2O、0.025 mg/L CoCl·6H2O、27.8 mg/L FeSO4·7H2O、37.3
mg/L Na2-EDTA；Vitamin, the mg/L of inositol 100,0.5 mg/L, L- glycine of nicotinic acid 2,0.5 mg/L of mg/L, VB6,
VB1 0.1 mg/L。
Step 6：By 12-15d or so steps（5）Rooting induction, regeneration plant is grown after 4-6 bar roots, by triangular flask
Bottleneck is opened, and is placed under natural conditions（At shady and cool, air circulation）Hardening, took out regrowth after 7-8 days from triangular flask, warp
Flowing water rinses out the culture medium residue in foundation portion, then, 6-8 is soaked with the carbendazim of volumetric concentration 0.1% with distilled water flushing several times
Min, goes in transplanting medium, outer button shading plastics, pours permeable 1-3 times daily, cultivates 2 DEG C of 28 scholar of temperature, evening minimum temperature
It is not less than 20 DEG C.Treat that seedling grows 3-4 piece young leaves, height of seedling 8-12cm or so is transplanted under normal soil in natural environment relaying
It is continuous to cultivate.The transplanting medium is that pine bark, peat soil, liver moss living and haydite press 1：1：1：1 mixing gained.The normal soil
For outdoor random acquired planting soil.Transplanted seedling/whole the transplanted seedling for survival rate=survive.The average transplant survival of 3 batch experiments
Rate is 86.3%, is shown in Table 3：
Using a kind of method that can obtain a large amount of Dracocephalum moldavica regeneration plants in a short time for providing of the present invention, with Dracocephalum moldavica without
The cotyledon of vaccine as explant carry out callus induction, inductivity is 91.2%, rise in value after callus squamous subculture compared with
Hurry up, differentiation degree is high, and differentiation rate is 84.2%, after the aftergrowth obtained after culture of rootage is transplanted survival rate to 86.3%,
A large amount of Dracocephalum moldavica regeneration plants can be obtained in a short time using this method, and good kind is provided further to expand cultivation Dracocephalum moldavica
Seedling can carry out the research such as genetic transformation of the species as outstanding scientific research material.
Claims (2)
1. a kind of method that anaesthetic Dracocephalum moldavica Tissue Culture Regeneration System is set up, it is characterised in that comprise the following steps：
1）The surface of the seed is sterilized and Aseptic seedling culture
The Dracocephalum moldavica seed of full seed is selected, rinsing 4-7min through flowing water removes surface dirt debris, aseptic water washing 4-7 times
After be placed on superclean bench, with volumetric concentration be 70% ethanol soak 30-40s, aseptic water washing 2-4 times；Volumetric concentration is used again
0.1%HgCl24-6min is sterilized, sterilized water concussion is rinsed 4-6 times, is seeded in the MS containing 100mL0Solid medium triangular flask
In, triangular flask body is wrapped up with brown paper, cultivated under the conditions of 28 ± 2 DEG C, after removal brown paper illumination cultivation after seed sprouting,
Illumination 12-14h/d, intensity of illumination 1800-2200 Lux；
2）The induction of callus：The Dracocephalum moldavica aseptic seedling of robust growth is taken, young tender cotyledon is cut and is inoculated in inducing culture MS1
On, every bottle of inoculation 3-4 block is transferred under normal illumination under no light after culture 9-12d and cultivated, observed after 13-16d and count callus
Inductivity is organized, induction illumination cultivation condition is：Illumination 12-14h/d, intensity of illumination 1800-2200 Lux, 28 ± 2 DEG C of temperature；
3）The squamous subculture of callus：Obtained yellow green callus will be induced to be seeded in subculture medium MS2It is middle carry out after
It is commissioned to train foster；Squamous subculture condition is：Illumination 12-14h/d, intensity of illumination 1800-2200 Lux, 28 ± 2 DEG C of temperature；
4）The induction differentiation of adventitious bud：It is indefinite by being gone to by the yellow green callus obtained after continuous squamous subculture 3-4 times
Bud differential medium MS3Middle to be induced, condition of culture is：Illumination 12-14h/d, intensity of illumination 1800-2200 Lux, temperature
28±2℃；
5）The root induction：Root induction culture medium MS is transferred to when obtaining regeneration bud 2-3 pieces by adventitious bud inducing4Given birth to
Root induction, condition of culture is：Illumination 12-14h/d, intensity of illumination 1800-2200 Lux, 28 ± 2 DEG C of temperature；
6）Transplantation of Regenerated Plantlets：Regeneration plant is grown after 4-6 bar roots, and triangular flask bottleneck is opened, and is placed at shady and cool, air circulation
Hardening, took out regrowth after 7-8 days, and the culture medium residue in foundation portion is rinsed out through flowing water, then, with distilled water flushing several times
It is 0.08-0.13% carbendazim immersion 6-8min with volumetric concentration, goes in transplanting medium, matrix is pine bark, peat soil, work
Liver moss and haydite, in mass ratio 1：1：1：1 mixing, outer button shading plastics, pours permeable 1-3 times, cultivates 28 ± 2 DEG C of temperature daily,
Evening temperature treats that seedling grows 3-4 piece young leaves, height of seedling 8-12cm is transplanted under normal soil in natural environment more than 20 DEG C
Continue to cultivate；
The MS0Solid medium is addition sucrose 30 g/L, agar 8-10 g/L, before sterilizing based on basic MS culture medium
PH is adjusted to 5.8-6.0；
The inducing culture MS1Using MS as minimal medium, to add 6-BA 0.5mg/L, 2,4-D 1.0mg/L, NAA
0.5mg/L, sucrose 30mg/L, agar 10mg/L, pH5.8-6.0；
The subculture medium MS2Using MS as minimal medium, to add 6-BA 0.5mg/L, 2,4-D 0.5mg/L, NAA
0.5mg/L, sucrose 30mg/L, agar 10mg/L, pH5.8-6.0；
The adventitious buds differentiation culture medium MS3Using MS as minimal medium, to add exogenous hormone 6-BA 0.8mg/L, NAA
0.1mg/L, sucrose 30mg/L, agar 10mg/L, pH5.8-6.0；
The root media MS4For using 1/2MS as minimal medium, addition exogenous hormone IBA 0.2mg/L, sucrose 30mg/L,
Agar 6mg/L, pH5.8-6.0；
The 1/2MS culture mediums are to halve a great number of elements.
2. the method that a kind of anaesthetic Dracocephalum moldavica Tissue Culture Regeneration System according to claim 1 is set up, it is characterised in that
The normal soil is outdoor random acquired planting soil.
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